Novel method of diagnosis of schistosomiasis and reagents therefore

ABSTRACT

A globinolytic enzyme has been isolated from Schistosome worms, purified, and characterized. This enzyme, either per se, or in the presence of stabilizing proteinaceous material, causes a rapid, histamine-like reaction when introduced into the dermal layer of mammals infested with Schistosomes. The enzyme itself has a molecular weight of about 27,000 and contains approximately 250 amino acid units.

United States Patent [191 Senft [451 Jan. 14,1975

[ NOVEL METHOD OF DIAGNOSIS OF SCHISTOSOMIASIS AND REAGENTS THEREFORE [75] Inventor: Alfred W. Senft, Providence, RI.

[73] Assignee: Research Corporation, New York,

22 Filed: Mar. 21, 1972 21 Appl. No.: 236,617

[52] U.S. Cl 195/62, 195/66 R, l95/l03.5 R,

424/12, 424/88, 424/9 [51] Int. Cl. C07g 7/026, A6lk 19/00 [58] Field of Search 195/65, 66, 62

[56] References Cited OTHER PUBLICATIONS Sawada et al., Japanese Journal of Exp. Med., Vol. 39,

No. 4, p. 339-345, (1969). Sato et 211., Japanese Journal of Exp. Med., Vol. 39, No.4, p. 346-353, (1969).

Primary Examiner-Lionel M. Shapiro Attorney, Agent, or FirmOmri M. Behr [57] ABSTRACT 27 Claims, 1 Drawing Figure NOVEL METHOD OF DIAGNOSIS OF SCHISTOSOMIASIS AND REAGENTS THEREFORE DESCRIPTION OF THE PRIOR ART The disease of Schistosomiasis is caused by fresh 5 water parasites of the Genus Schistosoma. Among the principal species causing the disease are S. mansoni, S. hematobium, and S. japonicum. These species have somewhat different geographic locations. It is estimated that the order of 250 million persons in Egypt, Africa, South America, The Phillipines, and certain areas of Southeast Asia, are infested by one of these species of Schistosoma. Related species, S. bovis, S. rhodaini and S. intercolatum are also known, but usually affect cattle rather than humans, although human infection from these species is not uncommon.

The known methods of diagnosis of this disease fall into two broad categories, namely the macro tests and the micro tests. The macro tests comprise the examina- 2 tion of urine or stools for the presence of worms, worm eggs, or recognizable worm debris. While the macro tests are generally speaking, reliable where a high degree of infestation is present, a negative reading in a macro test is no guarantee of non-infestation. Furthermore, the tests are time consuming and require the assistance of patient and highly skilled personnel. They are thus not suitable for the mass testing of population groups, particularly since the disease is endemic in those areas of the world where personnel of even minimal medical training are of highly limited availability.

The micro tests are principally immunological tests. These tests depend upon the basic theory that infestation with any organism will probably cause the formation of various types of antibodies, the presence of which may be detected by the introduction of an antigen to at least one of said antibodies thus causing an observable immunological response either in vivo or in vitro.

As early as 1927, a homogenate derived from adult worms of S. mansoni was utilized as test substrate to elicit an immunological response. It willbe recognized bythose skilled in the art that the use of a crude homogenate gives rise to many problems. The first problem is that the homogenate certainly contains many factors in addition to the possible antigenic factor by means of which it is desired to elicit a positive response. Hence, immunological reactions will occur which are denominated as false positives." Furthermore, the homogenate itself may contain factors which are harmful to the subject to which they are applied.

Efforts to minimize the problems attached to the crude homogenate led to the development of a delipidized aqueous extract of the adult worm generally designated as the Kagan antigen which has been utilized as the World Health Organization standard for screening in the field for about the last years. This length of use has established certain well recognized advantages and disadvantages of the Kagan antigen.

. The use of the Kagan substrate will give rise to a. positive reaction with satisfactory rapidity (circa 10-15minutes after intradermal injection) with adults who have been exposed to Schistosomiasis for some time. It does not appear to matter greatly whether the substrate utilized is an oridinary delipidized aqueous extract of the Schistosome worm or whether merely the total worm homogenate is utilized.

The Kagan antigen will not usually detect Schistosomiasis in children and young adults with any degree of reliability. Some reports show a range of from 30 to 60% false-negatives.

The Kagan serum will give rise to unacceptably high (circa reading of false positives, presumably due to immunological reactions caused by the portions of the substrate other than the desired antigen itself. A further disadvantage of the Kagan antigen, presumably 10 due to the same cause, is the appearance of secondary histaminic reactions or the maintenance of the initial histaminic reaction for between 24 to 48 hours after injection of the substrate.

Yet another problem associated with the Kaganantb 5 gen, is that a positive histaminic reaction will occur in a subject even after the subject has been cured of the disease when he has suffered infestation for a considerable amount of time. It has therefore been deemed desirable to seek an al- 0 ternate screening antigen which maintains the advantages listed above with few of the disadvantages associated with the Kagan substrateln particular, it has been deemed desirable to produce an antigenic substrate which will substantially eliminate the after-reaction, Le.

25 wherein the inflammation will substantially disappear within about 2 hours after initiation, and further, which will substantially reduce or indeed eliminate falsepositive readings.

In conjunction with this desired end, it is deemed helpful to isolate, purify, and ifpossible, elucidate the structure of the active substituent of the Schistosomi-.

Exp. Med. 39, 339, (1969)) These studies disclose the isolation of an antigenic proteinaceous material. However, the molecular weight and the isoelectric point of this material (or more properly, group of materials) are clearly entirely different from the material isolated and described in the invention disclosed herein.

' SUMMARY OF THE INVENTION In the process of the present invention a globinolytic enzyme of proteinaceous character is isolated from adult worms of the genus Schistosoma, in particular, from adult. worms of species Schistusvma mansoni.

In the process of the present invention adult worms are washed, and lyophilized. the lyophilizate is then taken up in a buffer, homogenized, and centrifuged in order to remove the undesired debris and non-soluble components. The supernate resulting from the centrifugation is then concentrated by passing a substantial proportion thereof through a 10,000 molecular weight exclusion gel filter and retaining the residue. The residue is then further purified suitably by chromatography upon dextran gel. The elution is followed by observing I the optical absorbance of the eluate at a predetermined wave-length and the fraction falling between the first and third absorption maxima is isolated. This fraction or fractions contain the biologically active antigenic material which, may be concentrated or dried by removal of the aqueous solvent.

The concentrate thus produced may be taken up in a suitable pharmacologically acceptable diluent for use as a diagnostic substrate. While the concentration of protein required. to elicit a histaminic reaction in a mammal infected with Schistosomiasis will clearly vary in accordance with the severity of the infestation, and the length of time during which the infestation has been present, the amount of injected material per dose should not be less than 300 nanograms.

The concentrate referred to herein may be further purified into 2 fractions, a so-called companion protein and the physiologically active antigen fraction. It has been found suitable to carry out this fractionation by chromatography on a phenylalanine-agarose column eluted with an eluent of pH between 3.5 and 4. Such elution will remove the companion protein from the column but will not remove the active fraction therefrom.

Elution with an eluent of pH less than about 3.0 or greater than about 4.5 will rapidly elute the active fragment. This active fraction has a specific activity of approximately twice that of the unfractionated material and has been substantially identified in a manner discussed in detail hereinbelow. Nevertheless, despite the enhanced activity of this material it does not possess the stability of the unfractionated material containing both the companion protein and the active fraction. The companion protein has mild antigenic activity and is believed responsible for the small delayed histaminic response of the novel antigen. Hence, the analysis of this material is of extreme interest and importance in I elucidating the structure of the antigenic material itself.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The starting materials of the process of the present invention are fresh worms of the genus Schistosoma, for example, S. mansoni, S. japonicum, or S. haematobium, suitably S. mansoni. These worms are obtained by perfusion of blood of infected white mice. This perfusion technique is well known to those skilled in the art. In the preferred procedure the worms are placed in shallow dishes and washed free of adherent blood cells and plasma using cool, citrated saline, or the like. The washed worms may then be either homogenized, or lyophilized. Since lyophilized worm material has been found to be quite stable it is preferred to carry out an initial lyophilization step. The washed worms are transferred to lyophilizate tubes, snap frozen and lyophilized at 40 to -80C. While it is preferred to keep the lyophilized material at about 125C in a super cold deep freeze before further processing it has been found that the material is substantially unaffected by storage at room temperature for several days.

The lyophilizate, or freshly washed worms, is then homogenized. It is customary to take up about 100 mg. of worms in from about 5 to about 20, suitably about 10 ml. of a suitable carrier. It is generally preferred to utilize a buffer having a pH of the same general range as the homogenate itself, i.e. between 3.0 and 4.5, it is preferred to use a buffer having a pH between 3.5 and 4, especially suitable are phosphate, acetate or citrate buffers of 0.1-0.3, suitably 0.2M concentration, however, the actual constitution of this buffer is not considered to be critical.

The homogenate is then centrifuged to remove the macro debris. The time and speed at which the centrifugation occurs should not be regarded as critical, however, it has been found satisfactory to centrifuge at between 10 to 25,000 g. for from about 45 to about 15 minutes. It is especially preferred to centrifuge for about minutes at 15,000 g. The supernate from the centrifugation is removed and filtered through a gel membrane. It is preferred to utilize a gel membrane having a pore size which excludes substances of 10,000 molecular weight. A convenient membrane to use is the Amicon P-lO. The preferred method of causing the supernate to pass through the membrane is to agitate the supernate within the filter unit above the membrane, preferably by means of a magnetic stirrer or the like, and applying to the upper surface of said supernate positive gas pressure utilizing a substantially inert gas such as nitrogen, helium or the like. It is generally preferred to utilize a positive pressure of between about 40 and about pounds per square inch. Filtration is permitted to proceed until approximately of the volume of the supernate has passed through the filter. This degree of filtration is not critical, however, it has been found that the filtrate at this point contains substantially no enzymic or immunological activity as defined hereinbelow, but merely contains free amino acids and some small peptides. This degree of concentration is useful since it facilitates application of the sample for the next step of purification.

The concentrated supernate is then removed from the upper surface of the gel filter and layered upon a gel column. It is preferred to use a cross linked carbohydrate gel, suitably a dextran, preferably cross linked with epichlorhydrin or the like.

The exact type of gel utilized is not critical provided that the largest molecule which passes with the solvent front would exceed 30,000 M.W. (for Sephadex G-200, this size is 200,000 M.W.). However, in order to achieve good separation of the components it has been found convenient to use Sephadex G-200.

It is especially preferred to carry out the chromatography described immediately hereunder at reduced temperatures preferably from about '5 to +5C. This may be done by carrying out the entire process in a cold room or in a refrigerated chromatographic column of the type which are commercially available. The column is eluted with a buffer of pH between 3.5 and 4, most suitably between 3.85 and 3.95, such as a phosphate, citrate or acetate buffer.

It has been found that separation of the fractions will proceed most efficiently if the reservoir of the elution buffer is so placed in relation to the gel column that there is very little pressure on the column due to the head of liquid in the reservoir.

It is preferred to follow the course of the chromatogram by observation of the optical density at a given wave length. In accordance with generally accepted practice in the protein field, the optical density at 280nm is observed and plotted relative to fraction number.

In the normal course of chromatography, the entire elutable charge on the chromatographic column is eluted in approximately 1 to 1.5 column volume. Three principal peaks are observed in surveying the plot of optical density against volume eluted. A first peak appear at approximately 0.5 column volumes, a second, minor peak, appear at about 0.8 column volumes and a third, major peak, appear at about 1 column volume. It should be noted that the exact location of these peaks and their distinctness is a necessary function of the activity of the adsorbant used in the column and the speed of elution. However, the general relationship of these peaks will remain regardless of the particular packing density of the chromatographic substrate and the speed of elution.

The first fraction has been found to have no immunological activity as defined hereinbelow. The third fraction contains inactive peptides derived from the worms or derived from residual globin within the worms. F raction 2 contains the factor having immunological activity. Fraction 2 may be isolated by evaporation of the solvent, for example, by lyophilization, freeze drying, evaporation at atmospheric pressure by passage of an inert gas thereover, evaporation at reduced pressure above the freezing point of the mixture, but below about 50C or any other similar means, especially preferred however is lyophilization. This is the fraction which is utilized in the immunological tests described hereinbelow. Since the inorganic material with which it is associated, i.e. phosphate citrate or acetate or the like is totally compatible with the human matabolic system, the lyophilizate may be utilized without separation therefrom. Such separation could of course be achieved by dialysis however, no known reason exists for such a further purification step.

An alternate mode of isolation of the proteinaceous material is by precipitation with a lower, i.e. C -C alkanol, suitably ethanol, iso-propanol and the like. The exact proportion of alkanol required is not critical, however where the alcohol/water proportion is less than 3:1 yields are unsatisfactory. This method is only recommended where it is desired to obtain a substrate substantially free of inorganic material, even at the price of lower yields.

The precipitated protein is separated by centrifugation in the usual manner and dried, suitably under reduced pressure, preferably at temperatures below about C.

It is however possible to separate the proteinaceous fraction into the so-called companion protein and the highly active antigenic enzyme. The separation may be carried out by chromatography on a phenylalanine/agarose column.

Elution of the column with a buffer having a pH of about 3.5 to about 4, that is to say, a buffer similar to that utilized for elution of the dextran gel column in the foregoing chromatographic step, yields a protein material which has substantially no enzymic, and rather low immunologic activity. This material is eluted rather rapidly, and accounts for approximately 50% by weight of the charge on the phenylalanine/agarose column measured in terms of peptide material. This material is designated as the companion protein.

The remaining fraction may be eluted by shifting the pH of the buffer either below 3.0 but 2.0, or above 4.5 but below 5.5. These figures are necessarily somewhat approximate, however, it has been found that this is the minimal shift required to achieve sharp elution of the remaining fraction without introducting shifts in pH substantial enough to cause chemical changes such as denaturing, to occur in the remaining fraction.

It is especially preferred to elute the column with dilute aqueous non-oxidizing organic or inorganic acids, for example, acetic, formic, mono-, diand trichlor or fluoracetic, or hydrochloric acids or the like, suitably dilute aqueous acetic or formic acid, preferably 0.2 molar aqueous acetic acid. The remaining fraction, designated as fraction 28 is rapidly eluted and may be isolated by reduced pressure evaporation. Fraction 2B is a globinolytic antigenic enzyme which causes strong histamine reaction in tissues containing antibodies due to the presence of Schistosomes, especially S. mansoni.

While fraction 28 may not be practically used in this 7 form it may be stabilized by means of stabilizing agents well known to the protein art. Among these agents may be included the liquid, pharmaceutically acceptable polyhydric alcohols, especially glycerol. Suitably, the fraction 28 enzyme is dissolved in glycerol as, say a l-l0% by weight solution, though this is not critical, and further diluted with an injectable diluent, of the type set forth below, shortly prior to use.

In an alternate though not necessarily especially preferred mode of isolation, material corresponding substantially to fraction 2 activity may be isolated by alcohol fractionation.

In this mode, lyophilized worms are homogenized suitably in a buffer of pH from about 3.0 to 4.0, such as an acetate, citrate or phosphate buffer, preferably in a 0.04M phosphate buffer at pH 3.0 and centrifuged for from about 20 to about 40 minutes at between 25,000 and 35,000 g. at from about +5 to about 5C.

The supernatant fraction obtained in this centrifugation is then subjected to alcohol fractionation, any lower (i.e. C -C alkanol may be used.

In the preferred mode, the supernate is held at a reduced temperature, suitably from about 0C to about +5C and there is added thereto an equal volume of l00% ethyl alcohol. After stirring for an additional period, say for about 10 to about 20 minutes, the precipitate thus formed is removed by moderate centrifugation at the same temperature range. It has been found suitable to centrifuge at between 2,000 and 3,000 rpm for about 3 to about lO minutes. The resultant super-. nate is separated in the usual manner, similarly cooled and a volume of ethyl alcohol equal to that in the supernate fraction is added to said supernate fraction and the precipitate collected by centrifugation in a similar manner. If desired, the precipitated material is collected, and if further desired, dissolved in a suitable buffer, for example, a 0.04M phosphate buffer of pH 3.0.

The purified antigenic material obtained by this method represents a recovery of appoximately 10% by weight of the protein content of the centrifugation supernate in contrast to approximately 30% recovery of the fraction 2 material calculated on the same basis. Moreover, the material isolated by this procedure is not as pure as the fraction 2 material. Nevertheless, it may be used for diagnostic testing purposes.

SCREENING METHODS In the preferred method of screening suspected subjects for infestation with Schistosomiasis, there is prepared a solution of fraction 2 material in a pharmaceutically acceptable injection medium. The media of choice are sterile water and Cocas bicarbonate/merthiolate diluent. The actual pH of the carrier utilized. is not of critical significance of all purposes of testing since upon injection the controlling pH will be the pH of the body fluids at that point.

The preferred diagnostic solutions comprise between 0.5 and 2.0 mg. of the protein component of the fraction 2 lyophilizate in 50 ml. of diluent. It is preferred to inject between 0.03 and 0.05 ml. containing from about 300 to about 1,000 nanograms of the diagnostic medium into the dermal layer of the subject. For purposes of comparison it is desirable to inject a similar quantity of the diluent used into an adjacent portion of the dermal layer in order to guard against false readings due to reactions caused by the introduction of foreign matter into the dermal layer rather than the true antigen antibody histamine-like response sought in the case of an infected person.

The diagnostic solution and a similar volume of diluent are injected into the dermal layer of the test sub ject. There is used about 300 to about 1,000 nanograms/test of fraction 2 material. Injection is made into any suitably exposed body area. Arms and shoulder blades are particularly suitable. The reaction is observed from about 10 to about 30 minutes after injection. In most cases infested subjects will show a substantial wheal where the fraction 2 material is injected after about 15 minutes. The diluent injection is used as a control to monitor allergic reactions not due to Schistosome infestation.

EXPERIMENTAL Example I Isolation of the Antigen S. mansoni worms are obtained by perfusion of infected white mice. The worms thus obtained are placed in shallow dishes and washed free of blood cells and plasma with sterile saline, and the washed worms transferred with lyophilizate tubes and snap frozen by insertion of the tubes into liquid nitrogen. The tubes are then allowed to warm to dry ice/methanol temperatures and are lyophilized at reduced pressure in a cold room. If desired, the material thus obtained may be stored at l25C.

The lyophilizate (100 mg.) is then homogenized at a bath temperature of circa C in 10 ml. of sodium acetate buffer (0.2M, pH 3.94). The homogenate is then spun at 15,000 rpm for 30 minutes (circa 15,000 g.). The supernate is then transferred to the upper chamber of a'filter unit fitted with a gel membrane (Amicon model 12 filter unit, P-l0 gel membrane, 0 to +C). The supernate is agitated gently by means of the integral magnetic stirrer and nitrogen pressure at ca. 60 psi. is applied to the upper surface of the supernate until the said supernate is concentrated to approximately to 1 ml.

The supernate is removed from the filter cell and layered upon a dextran gel, refrigerated column. (0.9 cm. X 36-40 cm. Kontes refrigerated column; Sephadex G-200 gel).

The column is eluted with sodium citrate buffer (pH 3.94). The reservoir is so set as to transfer minimal head pressure to the gel column. The fractions are collected in ca. 0.5 ml. aliquots using an automatic fraction collector. Elution is complete in from between 17 to 24 hours. The optical density of the fraction is measured at 280mm. A typical plot of absorbance against eluted volume is shown as the Figure.

Fractions 1 and 3 are not of immunological interest. The aliquots containing fraction 2 are collected, combined, and lyophilized to yield the antigenic material referred to herein as fraction 2. This material is the material utilized in the immunological work disclosed herein.

In accordance with the foregoing procedure, there may be utilized S. haematobium, S. japonicum, S. bovis, S. rhodhaini or S. intercolatum worms.

EXAMPLE ll Further Purification of Fraction 2 1.0 mg. of lyophilized fraction 2 is taken up in 0.5

ml. of water and layered on a phenylolanine/agarose column (Miles Yeda; 0.9 cm. X 5 cm.). The column is eluted with sodium acetate buffer (0.2M, pH 3.94). The course of the elution is followed by measurement of the optical density at 280nm. Only one fraction is eluted. The aliquots containing this fraction are combined and lyophilized, yield the so-callcd companion protein designated as fraction 2A.

The column is then eluted with dilute aqueous acetic acid (0.2M, pH 3.0). A second fraction is rapidly eluted, the aliquots containing said fraction arccombined and the eluent removed by evaporation. The fraction 2B protein is highly active immunologically but is labile and will be denatured upon freezing or thawing.

Analysis of Fraction 2B Enzyme a. Molecular Weight Measurement The initial filtration through the Biogel-P-lO membrane indicates that the enzyme contains no monomers smaller than mw 10,000. A calibrated dextran column was charged with laboratory standards of Ribonucleasc A, Chymotrypsinogen A, Ovalbumen, and Hemoglobin, in order to obtain a plot of elution volume against molecular weight to give a straight-line plot. Fraction 2B was run on this column and found to give a molecular weight of approximately 27,000 l,()00. (Gelclectrophoresis, using a polyacrylamidc get technique and sodium dodeeyl sulfonate (SDS dispersion agent, a molecular weight of ca. 28.000 has been determined).

b. Isoelectric Point Fraction 2B is run on an LKB ampholine column having an internal pH gradient of 3.0 to 10.0. The isoelectric point of fraction 28 is 3.55. The isoelectric point of the companion protein" by the same method is approximately 5.].

Spectral characteristics E pH 3.95: 24.2 X 10 It M"cm, E pH 13 37.5 X 10 it M"cm.

Amino Acid Analysis Amino Acid Residues/Mole enzyme Nearest Integer Lysine Histidine Argenine Tryptophan Aspartic Acid Threonine Serine Glutamic Acid Praline Glycine Alanine A Cystine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine Spectropholumelric Determination Extrapolated to time 0 of hydrolysis Value of hours hydrolysis Estimated error 3 15 residues Immunological Testing The activity is determined by dissolving 10 mg. of globin (prepared by the method of Kistler, et al., Helv. Chim./1cm, 36, 1058, (1953)) in 10 ml. of water and adding thereto a protein test solution having ca. 0.1 optical density at 280mm. The mixture is incubated for 60 minutes at 37C, the digestion of the globin terminated by the addition of ca. 5 drops of trichloracetic acid, whereby the globin is precipitated, the mixture centrifuged at ca. 2,000 rpm for 15 minutes at 40C to remove the precipitate and the optical density determined at 280nm to measure the quantity of small peptides remaining-in solution.

A preliminary survey of the relative activity of the material at various stages of purification, as measured by the optical absorbance at 280mm is given in Table 1 below.

TABLE I fected with Schistosomiasis raises an immediate histaminic response which reaches its peak in a few minutes and has substantially disappeared in a few hours.

It has been found that the injection of fraction 2A in similar concentrations, into a similarly infected subject, gives rise to a mild delayed response after approximately 48 hours.

In order to provide a severe test of the diagnostic ability of a composition comprisingfraction 2 in a suit able carrier such as sterile water or Cocas bicarbonate/methiolate medium very small doses of the order of 0.030.05 ml. containing approximately 20 pig/ml. were utilized as the test medium on children of the age group of 9 to years of age in areas known to be generally infested with Schistosomiasis and in neighboring areas known to be free of Schistosomiasis. As stated hereinabove, it is well known that the detection Typical Enzyme Purification Obtained from 100 mgs. Dried Worms a unit of activity is defined as absorbance 1.0 at 280nm in mixtures incubated with the specific globin substrate for 60 minutes at 37C. 1 protein content ofthcse preparations was determined by the method of Luwry, et a1. using bovine albumin as a standard immunological tests have been carried out upon mice, guinea pigs, monkeys, and human beings, both infected with Schistosomiasis and free of the disease. The reaction caused by the intradermal injection of about 300 nanograms of fraction 2 at a concentration of from about 5 to about ug/ml. in a generally accepted injection carrier such as sterile water, sterile water containing benzyl alcohol or Cocas (bicarbonate/methiolate diluent) into an infected subject is compacted to that caused by the carrier itself.

It has further been found that the injection of 300ng.

of Schistosomiasis by immunologic means in persons infected by Schistosomiasis for less than 15 years and particularly children and young adults, is notoriously difficult. Thus tests demonstrate the efficacy of the diagnostic medium in less than optimal doses under the harshest of test conditions. The accuracy of the testing was monitored by the administration of the Kagan antigen, well known in the art, to the same individuals tested by administration of fraction 2 and also by an examination of stool samples from almost all the subjects. In interpreting Table 11 below summarizing the results of fraction 28 into a human su j t kn wn to be of the skin study tests, it should be borne in mind that TABLE 11 immediate Reactions-15 Minutes Delayed Reactions-48 Hours Adult S. masom' Enzyme Adult S. manxoni Enzyme Antigen Antigen Antigen Antigen Positives Positives Positives Positives Total No. No. No. No.

St. Lucia 80 30 37.5 21 26.2

9-15 years 79 29 36.7 7 9.1) St. Vincent 10-15 years 44 6 13.6 0 0 St. Lucia Stool Positive 34 21 61.8 19 55.9 19 9 5 14.5 Stool Negative 44 S 11.4 2 4.5 2 4 5 1 2.3 Immediate and/or Delayed St. Lucia Stool Positive 34 29 65.9 20 58.9 Stool Negative 44 11 25.0 3 6.8

delayed reactions may be listed as applying to subjects who had immediate reaction, that is to say, within about 15 minutes, as well as those who only showed delayed reactions. Interpretation of Table II It should also be noted that in accordance with the immunologic theory discussed hereinabove, a positive skin test is possible for a stool negative subject since that subject may have at one time been infected with Schistosomiasis and while the worm infestation has ceased, the antibodies still remain in the subjectss blood stream. Thus, it will be seen that with respect to the immediate reaction, the tests generated by fraction 2 read out as giving the correct diagnosis in approximately fewer cases than that in the case of the Kagan antigen, a difference being well within the scope of experimental error in screening tests of this type. Thus, from the point of view of immediate reaction the fraction 2 antigen is of the same order of efficacy on immediate reading for infected subjects as the Kagan antigen at the level tested.

It is of great significance however, to note that whereas the Kagan antigen showed 13% infected subjects in an area known to be totally free of Schistosomiasis, such false-positives were not shown by the fraction 2 antigen. Furthermore, it is of equal significance to note that while many of the subjects tested with the Kagan antigen showed delayed reactions 48 hours after testing, a much smaller number of those tested with the fraction 2 antigen showed such delayed reaction. Such delayed responses which were noted after injection of the enzyme were extremely mild.

I claim:

1. A process for isolating proteinaceous enzymetic material from Schistosomes which comprises the steps of:

a. lyophilizing S. mansoni worms,

b. homogenizing the lyophilizate of (a) in a buffer having a pH of between 3.0 and 4.5,

c. centrifuging the homogenate of (b) at 10 to 25 X d. concentrating the supernate of (c) by passing up 80-95% thereof through a gel filter having pore size with an exclusion of 10,000 M. W. under pressure of inert gas,

e. subjecting the residual supernate of (d) to dextran gel chromatography using an eluent of pH 3.5 to 4.5,

f. measuring the optical absorbance of the eluate at 280nm, and

g. isolating those fractions whose absorbance falls between the first and third principal absorption maxima of (e and f).

2. A process according to claim 1 comprising the additional step of evaporating the solvent from the, chromatographic eluate.

3. A process according to claim 2 comprising the ad-.

step (e) is Sephadex 6-200 and the eluent is a citrate buffer of pH 3.94. I I

6. A process according to claim 5 comprising the additional step of evaporating the solvent from the final 5 eluate produced.

7. A process according to claim 5 comprising the additional step of lyophilizing the chromatographic eluate.

8. A process according to claim 5 comprising the additional steps of:

h. adding to said eluate at least 3 volumes of ethanol or isopropanol per volume of eluate i. separating the precipitate formed in step (h) by centrifugation.

9. A process for the fractionation of the product of claim 1 which comprises chromatographing said product on a phenylalanine/agarose column and eluting with a buffer of pH 3.5 to 4 to yield a first fraction.

10. A process according to claim 9 comprising the additional step of evaporating the solvent from said eluted fraction.

11. A process of claim 9 which comprises the further step of elution with an eluent of pH 30.

12. A process according to claim 11 which comprises the further step of evaporating the solvent from the eluate produced, at a temperature below 50C and above the freezing point of said eluate.

13. A process according to claim 9 which comprises the further step of elution with an eluent of pH 4.5 5.5.

14. A process according to claim 13 which comprises the further step of evaporating the solvent from the eluate produced, at a temperature below 50C and above the freezing point of said eluate.

15. A process in accordance with claim 11 wherein the eluent is selected from the group consisting of aqueous, formic, acetic, mono-, di-, and trichloro or fluoroacetic or hydrochloric acids.

16. A process according to claim 15 wherein the eluent is 0.2M acetic acid.

17. A process according to claim 16 comprising the further step of evaporating the solvent from the eluate products, at a temperature below 50C and above the freezing point of said eluate.

18. A process according to claim 11 comprising the additional steps of:

h. adding to said eluate at least 3 volumes of a lower alkanol per volume of eluate.

i. separating the precipitate therefrom.

19. A method of isolating proteinaceous material from Schistosomes which comprises the steps of:

a. lyophilization of S. mansoni worms,

b. homogenizing said lyophilizate of (a) in a buffer having a pH of between 3.0 and 4.0, 55 c. centrifuging the homogenate of (b) at from about to about 35 X 10 g, d. adding to the supernate of (c) an equal volume of 100% lower alkanol, agitating said mixture and centrifuging same at from about 2,000 to about.

20. The product produced in accordance with claim 21. The product produced in accordance with claim 4.

22. The product produced in accordance with claim 6.

23. The product produced in accordance with claim 8.

24. The product produced in accordance with claim 9.

25. The product produced in accordance with claim 12.

26. The product produced in accordance with claim 18.

27. An globinolytic enzyme derived from S. Mansoni capable of causing an immediate temporary histaminic reaction of the dermal layer of a mammalian host infected with S. Mansoni which comprises substantially of a proteinaceous material having a molecular weight of 27,000 i 3,000, an isoelectric point of 3.55, E at pH 3.95: 24.2 X 1t M cm, E at pH 13: 37 X l0 It M cm, containing 246 i l5 amino acids of substantially the following mutual molar distribution:

Lysine l4 Histidine 5 Argenine 9 Tryplophan 4 Aspartic Acid 26 Threonine I6 Serine 24 Glutamic Acid 27 Proline l l Glycine 20 Alanine l8 1% Cystine 2 Valine l4 Methionine 5 lsoleucine l0 Leucine 2| Tyrosine l l Phenylalanine 9. 

2. A process according to claim 1 comprising the additional step of evaporating the solvent from the chromatographic eluate.
 3. A process according to claim 2 comprising the additional step of lyophilizing the chromatographic eluate.
 4. A process according to claim 1 comprising the additional steps of: h. adding to said eluate at least 3 volumes of a lower alkanol per volume of eluate, i. separating the precipitate therefrom.
 5. A process according to claim 1 wherein the buffer of step (b) is a citrate buffer of pH 3.93, the rate of centrifugation of step (c) is 15,000g., the dextran gel of step (e) is Sephadex G-200 and the eluent is a citrate buffer of pH 3.94.
 6. A process according to claim 5 comprising the additional step of evaporating the solvent from the final eluate produced.
 7. A process according to claim 5 comprising the additional step of lyophilizing the chromatographic eluate.
 8. A process according to claim 5 comprising the additional steps of: h. adding to said eluate at least 3 volumes of ethanol or isopropanol per volume of eluate i. separating the precipitate formed in step (h) by centrifugation.
 9. A process for the fractionation of the product of claim 1 which comprises chromatographing said product on a phenylalanine/agarose column and eluting with a buffer of pH 3.5 to 4 to yield a first fraction.
 10. A process according to claim 9 comprising the additional step of evaporating the solvent from said eluted fraction.
 11. A process of claim 9 which comprises the further step of elution with an eluent of pH <3.0.
 12. A process according to claim 11 which comprises the further step of evaporating the solvent from the eluate produced, at a temperature below 50*C and above the freezing point of said eluate.
 13. A process according to claim 9 which comprises the further step of elution with an eluent of pH < 4.5 < 5.5.
 14. A process according to claim 13 which comprises the further step of evaporating the solvent from the eluate produced, at a temperature below 50*C and above the freezing point of said eluate.
 15. A process in accordance with claim 11 wherein the eluent is selected from the group consisting of aqueous, formic, acetic, mono-, di-, and tri- chloro or fluoroacetic or hydrochloric acids.
 16. A process according to claim 15 wherein the eluent is 0.2M acetic acid.
 17. A process according to claim 16 comprising the further step of evaporating the solvent from the eluate products, at a temperature below 50*C and above the freezing point of said eluate.
 18. A process according to claim 11 compRising the additional steps of: h. adding to said eluate at least 3 volumes of a lower alkanol per volume of eluate. i. separating the precipitate therefrom.
 19. A method of isolating proteinaceous material from Schistosomes which comprises the steps of: a. lyophilization of S. mansoni worms, b. homogenizing said lyophilizate of (a) in a buffer having a pH of between 3.0 and 4.0, c. centrifuging the homogenate of (b) at from about 25 to about 35 X 103g, d. adding to the supernate of (c) an equal volume of 100% lower alkanol, agitating said mixture and centrifuging same at from about 2,000 to about 3,000 rpm, e. adding to the supernate of (d) an equal volume of 100% lower alkanol, agitating said mixture, and centrifuging at from about 2,000 to about 3,000 rpm, f. isolating the precipitate of the centrifugation of (e).
 20. The product produced in accordance with claim
 2. 21. The product produced in accordance with claim
 4. 22. The product produced in accordance with claim
 6. 23. The product produced in accordance with claim
 8. 24. The product produced in accordance with claim
 9. 25. The product produced in accordance with claim
 12. 26. The product produced in accordance with claim
 18. 27. An globinolytic enzyme derived from S. Mansoni capable of causing an immediate temporary histaminic reaction of the dermal layer of a mammalian host infected with S. Mansoni which comprises substantially of a proteinaceous material having a molecular weight of 27,000 + or - 3,000, an isoelectric point of 3.55, E280 at pH 3.95: 24.2 X 103 lt M 1 cm, E280 at pH 13: 37 X 103 lt M 1 cm, containing 246 + or - 15 amino acids of substantially the following mutual molar distribution: 